Antibiotics t-7545 and process of producing same

ABSTRACT

A NEW ANTIBIOTIC T-7545 (VALIDAMYCIN) IS PRODUCED BY CULTURING STRAINS OF MICROORGANISM BELONGING TO THE GENUS STREPTOMYCES. COMPOSITION CONTAINING THE ANTIBIOTICS ARE USEFUL FOR CONTROLLING DIEASES IN PLANTS.

Aug. 21, 1973 MOTOO SHIBATA 3,754,083

ANTIBIOTICS T-7545 AND PROCESS OF PRODUCING SAME Filed Oct. 28, 1969 2Sheets-Sheet 1 I m .5 K lfi j 5 LL? 2500 zoooaoomonoomowoomoomcnmo MOTOOSHIBATA,

TAKASHI IWASA, HIROICHI YAMAMOTO, MITSUKO ASAI and o? KOMEI MIZUNO, 8Inventors w.) aowvilwsuva 'Z w/d air/M Attorneys 1973 MOTOO SHIBATA ETAL3,754,083

ANTIBIOTICS T7545 AND PROCESS OF PRODUCING SAME Filed Oct. 28, 1969 2Sheets-Sheet 2 8 Q [L s Q 7 Q- Isa) lm IKD Ian "(1) WAVELENGTH IKX) WmI600 ZGIDIKD v MOTOO SHIBATA, TAKASHI IWASA, HIROICHI YAMAMOTO MITSUKOASAI and I gKomEl MIZUNO,

9 8 Inventors (/o) BQNVLIINSNVHL At tome United States Patent Olhce3,754,083 Patented Aug. 21, 1973 U.S. Cl. 424-118 4 Claims ABSTRACT OFTHE DISCLOSURE A new antibiotic T-7545 (Validamycin) is produced byculturing strains of microorganisms belonging to the genus streptomyces.Compositions containing the antibiotics are useful for controllingdiseases in plants.

The present invention relates to new antibiotics. More particularly theinvention provides new antibiotics T7545 (Validam'ycin) which is made upof antibiotic T7545A, antibiotic T7545-B esters thereof, salts thereofor the and is obtained by cultivating the antibiotic T-7545-producingstrain belonging to the Actinomycetes.

In search of new antibiotic substances, the present inventors isolated alarge number of soil microorganisms and studied their metabolites. Theresearch work led to the finding that certain soil microorganisms arecapable of producing a new antibiotic designated as T-7545, that thosemicroorganisms belong to the Actinomycetes and that it is possible toobtain said antibiotic by cultivating those microorganisms so that thesaid antibiotic may be accumulated in the culture medium. In the instantspecification, the term Antibiotic T-7545" ester thereof, salts thereofrefers to the Antibiotcs T-7545-A, T-7545-B or mixtures thereof.

Thus, the invention relates to a new antibiotic, which is produced bycultivating a T-7545-producing strain belonging to the Actinomycetes(hereinafter referred to sometimes as the T-754S-producing strain) sothat the said antibiotic is produced and accumulates in the fermentedbroth and recovering the antbiotic so accumulated from said fermentedbroth.

Furthermore, it is found unexpectedly that these new antibiotics T-7545show an excellent controlling etfect against plant diseases, forexample, the sheath blight of rice plant by the application in vivo,although they show no antimicrobial potency in vitro against bacteriaand fungi.

It is the principal object of this invention to provide new antibioticT-7545 and the method of preparing the same.

Another object is to provide a fungicide for combatting bacterial orfungal plant diseases showing no substantial phytotoxicity.

Another object is to provide a fungicide which is substantiallynon-toxic against both human beings and animals as well as fish.

'Further object is to provide a concentrate form of said fungicide,which is applicable, simply diluted at the use, to the host for the samepurpose as mentioned just above, and which is more stable and moreconvenient in storage or transport than the diluted composition for theready use to the plant.

Further obiect is to provide a systemic fungicide which shows a stronginfiltrating action to plants.

Other objects will be apparent from the description detailed hereinafterin this specification.

The antibiotics T-7545 is obtained by cultivation of a T-7545 producingstrain belonging to the Actinornycetes, so long as it is capable ofproducing the antibiotic T-7545. For example, the strain which wasisolated from the soil by the present inventors collected in AkashiCity, Hyogo Prefecture, Japan and named Streptomyces hygroscopicus var.limoneus, as well as its related strain, may be employed to particularadvantage.

Some of the microbiological and cultural characteristics of Streptomyceshygroscopicus var. limoneus are shown below. In the followingdescriptions of the cultural characteristics, Rdg. means the color nameaccording to Ridgeways Color Standard and Color Nomencla ture."

(l) Morphological characteristics The aerial mycelium of this strainshows monopodial branching and the conidia chain are formed in spiral.They are ovoid or rectangular, ranging from 1.0'1.3;t x 1.0-1.5,u, andhas a smooth surface.

(2) Growth cultural characteristics The following culturalcharacteristics, unless otherwise stated, are those observed uponcultivation at 28 C. Where cultivation was carried out at anytemperature other than 28 C., that temperature is indicated inparentheses.

(a) Czapeks agar Growth: Colorless, folded.

Reverse: Raw Sienna (Rdg., III, 17-i) to Sudan Brown (Rdg., HII, 15'-k).

Aerial mycelium: Tilleul 'Buif (Rdg., XL, 17"'-f) to Light Buff (Rdg.,XV, 17'-f), partially Mouse Gray (Dg., LI, 15"') along the periphery ofthe colony.

.Soluble pigment: Yellow with a faint brownish tinge.

(b) Glucose Czapeks a-gar Growth: Colorless to Sulphin Yellow (Rdg., IV,

21-i), folded.

Reverse: Raw Sienna.

Aerial mycelium: Tilleul Buff to Massicot Yellow (Rdg., XVI, 21'f),partially Light Olive Gray (Rdg., LI, 23'd) along the periphery of thecolony.

Soluble pigment: Yellow with a faint brownish tinge.

(c) Glycerin Czapeks agar Growth: Colorless to Orange Citrine (Rdg., IV,19-

k), folded.

Reverse: Raw Sienna.

Aerial mycelium: Tilleul Buff to Massicot Yellow,

partially Light Olive Gray.

Soluble pigment: Yellow with a faint brownish tinge.

(d) Glucose asparagine agar Growth: Colorless.

Reverse: Old Gold (Rdg., XVI, l9-i) to Antimony Yellow (Rdg., XV, l7b)to Cinnamon Brown (Rdg., XV, 15'k).

Aerial mycelium: Light Olive Gray to Mouse Gray,

with yellow patches and black moist areas.

Soluble pigment: Light Brown.

3 (e) Calcium malate agar Growth: Primuline Yellow (Rdg., XVI, 19')Reverse: Primuline Yellow. Aerial mycelium: Scarce at first, but TilleulBulf to Light Olive Gray later. Soluble pigment: Pale yellow. (f) Starchagar No growth. (g) Modified starch agar with the following componentsPercent Soluble starch 1 Potassium monohydrogen phosphate 0.3 Calciumcarbonate 0.3 Magnesium sulfate 0.1 Ammonium sulfate 0.2 Sodium chloride:05 Agar 2 Growth: Colorless to Barium Yellow (Rdg, XVI,

Reverse: Deep Colonial Buff (Rdg., XXX, 21"-b) to Snuff Brown (Rdg.,XXIX, l5"-k). Aerial mycelium: Cartridge Buff (Rdg., XXX, 19"- f) toMouse Gray, with black moist areas. Soluble pigment: Light Brown.

Hydrolyzation of starch Was observed. Tyrosine agar Growth: Colorless toStrontian Yellow (Rdg., XVI,

23'). Reverse: Pale Ochraceous Buff (Rdg., XV, f)

to Light Ochraceous Butt (Rdg., XV., l5'd). Aerial mycelium: None.Soluble pigment: None. (i) Yeast extract agar Growth: Colorless, folded.Reverse: Cream color (Rdg., XVI, 19-f). Aerial mycelium: White. Solu'blepigment: Light Brown. (j) Nutrient agar (37 C.)

Growth: Colorless. Reverse: Colorless. Aerial mycelium: None. Solublepigment: None. Glucose Nutrient agar (37 C.) Growth: Colorless,wrinkled. Reverse: Cartridge Buff to Pale Ochraceous Buff. Aerialmycelium: None. Soluble pigment: None. (1) Nutrient broth (37 C.)

Growth: Colorless surface growth, and colorless flocculent growth in thebottom. Aerial mycelium: None. Soluble pigment: None. (m) Glucosenutrient broth (37 C.)

Growth: Surface growth, Cartridge Bulf, and colorless fiocculent growthin the bottom. Aerial mycelium: None. Soluble pigment: None. Potato plugGrowth: Colorless to Pale Ochraceous Bulf. Aerial mycelium: Tilleul Bulfto Mouse Gray.

The plug turns to Sayal Brown (Rdg., XXIX,

15"-i). Carrot plug Growth: Colorless. Aerial mycelium: White to MouseGray.

The plug turns to Cinnamon Rufous (Rdg, XIV, 1l'-i) to Cinnamon Brown.Cellulose Growth: Chartreuse Yellow (Rdg., XXXI, "d)

to Reed Yellow (Rdg., XXX, 23"b). Aerial mycelium: Mouse Gray. Solublepigment: Pale Yellow. Gelatin (25 C.) Growth: Very poor. Aerialmycelium: None.

4 Soluble pigment: None.

Gelatine is liquefied slightly. The same is true with nutrient gelatin.Whole egg (37 C.) Growth: Colorless. Aerial mycelium: None. Solublepigment: None. Litmus milk (37 C.) Growth: Surface growth, Cream colorto Seashell Pink (Rdg, XIV, 11'-f). Aerial mycelium: None.

The medium is weakly coagulated, then, peptonized to turn to Army Brown(Rdg., XL, 13" 'i) and becomes weakly acidic.

- (t) Lalfers medium (37 C.)

Growth: Naples Yellow (Rdg., XVI, l9'-d) at first and Light Bull later.

Aerial mycelium: None.

Soluble pigment: None.

No liquefaction.

Peptone glucose agar Growth: Chartreuse Yellow.

Reverse: Honey Yellow.

Aerial mycelium: Thin, Cream Buif.

Soluble pigment: Yellow with a brownish tinge.

Plain Agar Growth: Scarce and colorless growth into substance of medium.

Reverse: Colorless to Mouse Gray.

Aerial mycelium: Scarce, Tilleul But]? to Mouse Gray.

Soluble pigment: None.

(3) Physiological properties (4) Utilization of carbon sources Table 1shows the utilization of carbon sources by the present strain asexamined by the method of Pridham et al. (Journal of Bacteriology 56107-114 (1948)).

TABLE 1 Utilzation of carbon sources by streptomyces hygroscopicus var.limoneus Erythritol Inositol. Adonitol D-mannitol- Sorbitol Duloito1D-xylose- Trehalose- L-arabmose Salicin L-sorbose--- Eseulin D-galactoseTnnlin Glucose Dextran D-fruot0se- Mannose L-rharnnose arch Mehbmse...Glycerol Maltese-.. Sodium acetate--- Sucrose- Sodium succlnate LaetoseSodium citrate Ratfinose Calcium 2-keto gluconate-.

N 0TE.-++ =Well utilized; Fairly utilized; Not utilized.

Thus, the present strain shows monopodial branching, the tip of itsaerial myceliurn being coiled. The conidia have a smooth surface. Itgives bright yellow to boilcolored growth on synthetic media, generally;produces no brown soluble pigment on protein-containing media.

The foregoing cultural characteristics were compared with thedescriptions given in Bergeys Manual of Determinative Bacteriology, 7thed. (The Williams and Wilkins, 1957), S. A. Waksmans. The Actinomycetes,vol. 2 (The Williams and Wilkins, 1962) and Ralph Hiitters Systematikder Streptomyceten (Es. Karugas, 1967), for instance. It was found thatthe strain resembles Streptomyces ambofaciens, Streptomyces platensisand Streptomyces hygroscopicus.

However, despite of the close resemblance in the color of bothvegetative and aerial mycelium, Streptomyces ambofaciens and the presentstrain differ from each other in that the former does not give blackmoist spots in the aerial mycelium, that it liquefies gelatin in amedium degree and gives yellow flocculent growth in the liquefied partand that it utilizes carbon sources different from those utilized by thepresent strain. Streptomyces platensis differentiates itself from thepresent strain in that the former produces a Deep Olive vegetativemycelium on Czapeks agar, with the reverse of the colony turning darkolive with the passage of time, that it gives cream to dull yellowishgrowth on starch agar with its aerial mycelium changing in color fromwhite to Mouse Gray with black patches, and that it utilizes carbonsources different from those utilized by the present strain. Comparisonof the descriptions of Streptomyces hygroscopicus with the culturalcharacteristics of the present strain shows that the present straindifierentiates itself from Streptomyces hygroscopicus in that the growthor the reverse of the colony of the present strain shows the lightyellow to buff on Czapeks agar (inclusive of glucose Czapeks agar andglycerol Czapeks agar), glucose asparagine agar, calcium malate agar andother media, and produces a yellowish white to yellow aerial mycelium onsaid Czapeks agar media. However, many of the cultural characteristicsof this strain coincide with the stable characteristics of Streptomyceshygroscopicus indicated by Tresner and Backus (Applied Microbiology,vol. 4, p. 243, 1956). Accordingly we identified the present strain as avariant of Streptomyces hygroscopz'cus and designated it as Streptomyceshydroscopicus var. limoneus. This strain has been deposited with theInstitute for Fermentation, Osaka, under the accession number ofIFO-12703.

The strain has been deposited with American Type Culture Collection(ATCC) under the Accession number ATCC 21431.

As a general trait of Actinomycetes, their microbiologicalcharacteristics are highly mutative and Streptomyces hygroscopicus var.limoneus is no exception to the rule. For example, its culturalcharacteristics and pattern of utilization of carbon sources aresusceptible to change, and there can be many mutants. Particularly, ofthis strain, mutants which have yellow aerial mycelia are easilyobtained, e.g., a mutant designated as 'IFO-12704 has similar propertiesto the strain IFO-12703 except having yellow aerial mycelia which doesnot become hygroscopic. In addition to the deposit with Institute forFermentation, Osaka, deposit of this mutant has also been made with ATCCunder accession No. ATCC 21432. However, even those mutants may beemployed in this invention in so far as they possess the capacity toproduce the T-7545 antibiotic. It does not matter, of course, if themutants are induced spontaneously or artificially. For purposes of thisinvention these mutants are the full equivalents of theabove-denominated strains.

In the culture medium employed in this invention, assimilable carbonsources, digestible nitrogen sources, inorganic salts and the like areincorporated. If required, there may be added trace elements such astrace nutrients, growth factors, precursors, etc. to the culture medium.The carbon sources which the T-7545-producing strain assimilatesinclude, among others, hydrocarbon, glucose, sucrose. molasses, starch,dextrin and glycerine. The nitrogen sources include such organicnitrogenous compounds such as meat extract, soybean flour, corn steepliquor peptone, casein, etc., as well as such inorganic nitrogencompounds as nitrates and ammonium compounds, and any of them can beemployed to advantage.

While cultivation can be carried out by surface culture, it is moreusual to adopt the aerobic submerged culture. In the case of thesubmerged culture, the pH of the medium is preferably near neutral, andwhile growth occurs at the incubation temperature of 20 to 40 C., it ispreferable to maintain the medium within the range of about 23 to 37 C.The accumulation of the objective antibiotic is complete in 4 to 7 days.

The T-7545 does not inhibit the growth of bacteria and fungi in vitrotest but only causes in Pellicularia sasakii and its closely relatedfungus an abnormal branching (excessive branching, or branched hyphaebecome an umbel like form) on the tips of the hyphae. Therefore, thebioassay of the present anitbiotic T-7545 is conducted in the followingmanner.

Pellicularia sasakii and plain agar are used as the test organism andthe assay medium respectively. The test organism is cultivated on apotato (sucrose) agar plate for 2 to 5 days, and the resulting cultureis used to inoculate at the center of a 9 cm. Petri dish plate ofmodified Pfeffers medium, which is then incubated at 27 C. for 2 days.By the end of this period, the mycelium will have spread over the entiresurface of the plate. The growth on the curcumference about 3 to 3.5 cm.in radius from the center is cut out with a cork borer and the agar discthus obtained is used as the inoculum.

A serial dilution series of agar plates containing varying concentrationof the antibiotic T7545 is prepared in the same manner as a conventionalagar dilution method.

A glass disc of 8 mm, in diameter and about 0.2 mm. thick, is placed inthe center of each of the aforementioned dilution series of agar plates,and the agar disc inoculum is then placed on the glass disc. Afterincubated at 27 C. for 40 hours, the result is evaluated. Naked-eyeexamination reveals that Pellicularia sasakii the tip of hyphae of testorganism growing from the agar disk inoculum undergoes abnormalbranching. The dilution unit means the value of maximum dilution inwhich the solution is able to show such abnormal branching. The aqueoussolution containing 1000 'y/ml. of purified T- 7545- A or T7545-B(purification described hereinafter) shows 100,000 dilution unit/ml. and1500 dilution unit/ ml., respectively.

Modified Pfeffers medium, which is employed in this assay, has thefollowing composition.

A chelate compound, iron sodium ethanol ethylenediamlne triacetate.

Before using the medium, the following vitamins are added in thespecified amounts.

(Weight per ml. of medium), 'y/rnl.

Thiamine Riboflavin 1 Calcium pantothenate 1 Niacin 1 Biotin 0.005 Folicacid 0.5 Pyridoxine hydrochloride 2 p-Aminobenzoic acid 0.5

Cyanocobalamine 0.0002

When Streptomyces hygroscopicus var. lim'oneus is cultivated in theabove manner, the antibiotic T-7545 is produced and accumulated mainlyin the liquid phase of the broth. Therefore, to recover the antibioticT-7545, it is preferable to filter the broth and, then, recover theantibiotic from the resulting filtrate. When the microorganism employedis capable of producing only one of the two antibiotics T-7545-A andT-7545-B, the produced antibiotic can, of course, be collected from thecultured broth in per se conventional manner. But, in case where themicroorganism employed can produce simultaneously both of them, they areconcomitantly accumulated in the broth, so that both antibiotics arecollectible from the broth as a mixture of them or as an individualantibiotic T-7545-A or T-7'545-B.

For the purpose of isolating the antibiotics, means which areconventionally used to recover the metabolites of microorganisms fromtheir broths can be employed either singly or in combination. Thus, themeans include such techniques as filtration, concentration, ion-exchangechromatography with ion exchangers, adsorption chromatography on activecarbon, silica gel, alumina, etc., gel filtration with Sephadex (tradename of Pharmacia), Bio- Gel-P (BIORAD Laboratories), etc., the use ofvarious solvents to bring the solute into another liquid phase,precipitation, removal of impurities, dialysis, drying andrecrystallization, among others.

For the separation and purification of antibiotic T-7545 fromimpurities, for example, its water soluble and basic properties areused, e.g., while water soluble lower molecular impurities are passedthrough, the antibiotic T-7545 can be adsorbed on active carbon andeluted with an acid aqueous alcohol and/or aqueous acetone.

The antibiotic T-7545 can be adsorbed strongly on cation exchange resinswhen it is in a state of neutral or weakly acid solution, and elutedwith bufferized basic solution, or an aqueous salt solution. Theantibiotic T-7545 can be adsorbed weakly also on anion exchange resinsand eluted with water.

The separation of antibiotics T-7545-A and T-7545-B is carried out byion exchange chromatography using bufferized cation and anion exchangeresins. For example, the antibiotic T-754S can be adsorbed on Dowex-SOW-X2 bufferized with the pyridine-acetic acid-bufi'er (pH 5-6), andT-7545-B is eluted with the bufierized solu tion (pH 6.2-6.8), while theT-7545-A is eluted with the bulferized solution (pH 7-7.5). When Dowex1-X2 is used, the antibiotic T-7545-A is first eluted, and then T-7545-Bis eluted with water. When silica gel is used as the adsorbent, theantibiotic T-7545-B is first eluated, then A follows withn-propanol-acetic acid-water (411:1).

Among the ion exchange resins (strong or weak) which can be employed forthe aforesaid purpose, there may be mentioned, for example, AmberliteIR-IOO, 112 and 120, Amberlite XE-69, Amberlite IRC-SO, Amberlite XE-89,Amberlite XE-64, Amberlite IR-45 and IRA-900 (all the above resins arethe trade names of Rohm and Haas, Co.). Dowex-50-X2, Dowex-l-XZ,Dowex-l-XS (These three resins are trade names of Dow Chemical, Co.),Duolite CS-6S (trade name of Chemical Process, Co.), Permutit-SO (tradename is Permutit, Co.), etc. These resins may be prepared by suchmethods as described in Ion Exchange Resin (Robert Kumin, Published byJohn Wiley & Sons, Inc., New York, N.Y., USA. pp. 87-97), or thosedescribed in literature references cited therein, and the proper meshsize and degree of cross-linking should be chosen for each resin-typeaccording to the particular phase or stage of the process.

The afore-mentioned salts not only include the alkali salts of strongacids such as sodium chloride and ammonium chloride but also the alkalisalts of organic acids such as sodium acetate, ammonium acetate, etc.,as Well as the salts of weak acids or weak bases such as those of aceticacid, pyridine and the like.

The desired substance which has been rather highly purified (purity orhigher) in the aforesaid manner is derived into its hydrochloride (1mole), which can be further purified by the crystallization with amixture of methanol and acetone as a white crystalline powder.Therefore, both the free form of the substance and its acid salt (e.g.hydrochloride, sulfate, organic sulfonate) may be used to advantage fortheir purification.

The antibiotics which can thus be obtained, have the following physical,chemical and physiological properties.

(1) Physical and chemical properties of the antibotic T-7545-A Ninhydrinreaction: faint purple color (d) pK'a and molecular weight pK'a 6.2 Themolecular weight estimated by titration 510125 (e) Ultravioletabsorption spectrum No characteristic absorption is observed at above210 mu. (f) Infrared absorption spectrum (FIG. 1)

Significant absorption bands 3450(8), 2900(M),

1640(M), 1450(M), 1410(M), 1370(M), 1160- (M), 1120-1000(8), 920(W),900(M), 855(M). The abbreviations M, W and S in parentheses denotemedium adsorptions, weak adsorptions and strong adsorptions,respectively. (g) The nuclear magnetic resonance spectrum of the samplesuggests the presence of methine protons adjacent to the oxygen. (h)Optical rotation i15 (C=1, H 0) 110:15 (C=1, pyridine) 92.5 i 10' (C: 1,dimethylformamide) (i) Elementary analysis Constituent elements: C, H, Nand 0. After drying with phosphorous pentoxide at 60 C. for 6 hoursunder reduced pressure, the sample still shows the presence of water. C,47.6-11.5 H, 7.17i0.5%; N, 3.01i0.5% (j) Paper chromatography Table 1shows the R values measured by paper chromatography and thin-layerchromatography on Whatman No. 1 papers and those measured by Whatman No.1 papers and those measured by thin-layer chromatography on silica gel.An alkaline potassium permanganate solution (an aqueous solutioncontaining 2% sodium carbonate and 1% potassium permanganate) is used asa detecting reagent.

TAB LE 1 Paper chromatography Thin-layer T-7545-A chromatoghydroraphy,Solvent system T-7545-A chloride T-7545-A n-Butanol: acetic acid: water(4:1:2) 0. -0. 12 0.10-0. 12 0.10 70% aqueous acetone 0. 44 0.37-0.440.53 n-Butauol:ethanolzwaterzconc.

aqueous ammonia (40:10:49:1) 0. 11-0. 12 0.11-0. 12 0. 08 Ethyl acetate:acetic acidzwater :1: 0.01 0. 01 0. 00 Eth lacetate: 'dinezwater(2?1:2).-.- 1 0. 01-0. 02 0.01-0.02 0. 01 n-Butanol:pyridine:water(4:2:1) 0.05 0.05 0.18 n-Butanol:pyridine:water (4 0. 46-0. 48 0. 46-0.48 0.27 80% phenol(NH3) 0. 55 0. 55 0.38 n-Butauol:ethanol:water;pyridine (35 :15:40:10) 0. 85-0. 39 0.35-0.39 0. 44n-Propanol:water:conc. aqueous ammonia (70:29:1) 0.06-0.07 0. 06 0- 07n-Propanol: acetic acidzwater (4: 1 :1) 0.

(k) Electrophoresis Paper electrophoresis was carried out using 0.05 Mboric acid buffer (pH 10) at a potential difference of 2 kv. for 2hours, and using pyridineacetic acid (pH 6.0) at a potential differenceof 2 kv. for 3 hours. The mobility spectra of the present substance areas follows.

Cm. Boric acid buffer (pH 10) +1.5 Pyridine-acetic acid buffer (pH 6.0)8.5

(1) Acid salt The antibiotic T-7545-A has the weakly basic property,forms a salt with acid (e.g. hydrochloric acid, sulfuric acid, organicsulfonic acids, etc.).

(2) Physical and chemical properties of the antibiotic (a) Appearance,M.P., etc.

A weakly basic white hygroscopic powder, which has no definite meltingpoint, and decomposes at 95-140 C.

(b) Solubility Readily soluble in water and polar organic solvents (e.g.methanol, pyridine, dimethylformamide, dimethylsulfoxide, etc.);sparingly soluble in acetone and ethanol; and insoluble in ethylacetate, ether and petroleum ether.

(0) Color reactions Molish reactions: positive Fehling reaction (underheating): positive Anthrone reaction: positive Phenol-sulfuric acidreaction: positive Orcinol-sulfuric acid reaction: positiveNaphthoresorcin-sulfuric acid reaction: positive -Benzidine-periodatereaction reaction: positive Peptide detection reagent (tertiarybutylhypochloride reaction: positive Alkaline potassium permanganate:reduce Ninhydrin' reaction: faint purple color.

(d) pK'a and Molecular weight The molecular weight estimated bytitration 520i25 (e) Ultraviolet absorption spectrum No characteristicabsorption is observed at above 210 m (f) Infrared absorption spectrum(FIG. 2).

Significant absorption bands 3400 (S), 2910 (M), 1638 (M), 1415 (M),1080 (S), 1025 (S), 900 (M), 840 (M), The abbreviations M, W and S inparentheses, denotes medium absorptions, weak adsorptions and strongadsorptions.

10 (g) The nuclear magnetic resonance spectrum of the sample suggeststhe presence of methine protons adjacent to the oxygen. (h) Opticalrotation [a] =l02:10 (C=1, H O) (i) Elementary analysis Constituentelements: C, H, N and 0. After drying with phosphorus pentoxide at 60 C.for 6 hours under reduced pressure, the sample still shows the presenceof water. C=46.46:1.5%

H=7.06i0.5% N=2.44:0.5%. (j) Thin-layer chromatography The Rf valuemeasured by thin-layer chromatography on silica gel in which an alkalinepotassium permanganate solution (an aqueous solution containing 2%sodium carbonate and 1% potassium permanganate) is used as a detectingreagent and n-propanolzacetic acid:water (4:1:1) is used as a developer,was 0.43. (k) Electrophoresis Paper electrophoresis was carried outusing 0.05 M boric acid butler (pH 10) at a potential difference of 2kv. for 2 hours, and using pyridine-acetic acid (pH 6.0) at a potentialdifference of 2 kv. for 3 hours. The mobility spectra of the presentsubstance are as follows.

Cm. Boric acid buffer (pH 10) +6.0 Pyridine-acetic acid buffer (pH 6.0)-4.0

(1) Acid salt The antibiotic T-7545-B has the weak base-property,

forms a salt with acid (e.g. hydrochloric acid, sulfuric acid, organicsulfonic acids, etc.).

(3) Biological properties of the antibiotic T-7545 Minimal inhibitoryconcentration, 7/1111.

Conditions of Assay organism assay T-7545-A 'I7545-B Pyricularia oryzae28 0., 40 hours-.. 100 Pellzcularia aasakii .do 100 100 Colletotrichumlagenariumd0. 100 100 Alternarz'a kikuchiamz .do 100 100 Aspergillusniger do 100 100 Penicillium chrysogenum- -do 100 100 f cerevz'sz'aa do100 100 Candida albicans do 100 100 Trichophyton mentagrophytes. ..-do100 100 Xanthomonas oryzae .do 100 100 Bacillus subtilis 37 (3., 14hours- 100 100 F" e aureus d 100 100 Sarcina Zutea 100 100 Escherichiacolzl. 100 100 Proteus vulgarz's. 100 100 Mycobacterium avium. 100 100Mycobacterium ATC C 607 100 100 Tables 3 and 4 show the activities ofthe present substances against several plant pathogenic fungi asmeasured by the method applying the same microbiological assay procedureas described before.

In the tables, each figure means the value of maximum dilution whichgives rise to abnormal branching by an aqueous solution (1 mg./ml.) ofthe purified antibiotic T-7545-A or T-7545-B respectively.

1 1 Table 3.Activities of T-7545-A against plant pathogenic fungi Thevalue of maximum dilution which gives rise to abnormal branching of theAssay organism: tip of hyphae Pellicularia sasakii 100,000 Rhizoctoniasolani 50,000 Corticium rolfsii 100 Phytophthora infestans 2000 Table4.Activities of T-7545-B against plant pathogenic fungi The value ofmaximum dilution which gives rise to abnormal branching of the Assayorganism tip of hyphae Pellicularia sasakii 1500 Rhizoctonia solam'Corticium rolfsii 100 Phytophrhora infestans 2000 Thus, although T-7545does not inhibit a number of microorganisms including even Pelliculariasasekii in ordinary test methods (in vitro), it showed remarkablecontrolling eifect against the sheath blight of rice plant in VIVO.

The ultra-violet region of the spectrum, infrared absorption spectrum,nuclear magnetic resonance spectrum, elementary analysis, and the otherchemical properties of the antibiotic T-7545 suggest that it is anaminocyclitol antibiotic containing glucose in the molecule. However,the present antibiotic differs from the known aminoeyclitol antibioticsor their related compounds in the starting microorganisms, the physicaland chemical properties as well as the physiological properties. Hencethe present antibiotic is considered to be a new antibiotic. T-7545-Aand T-7545-B have almost similar infrared absorption spectra andultra-violet absorption spectra, but Rf value of thin-layerchromatography, mobility of high voltage paper electrophoresis and pK'aare clearly difierent from each other and they are separable by theliquid chromatography.

Although these antibiotics show no substantial antimicrobial activity invitro, they have strong protective efiect on the living plants againstPellicularia sasakz'i, Leptosphaerz'a salvinii, Rhizoctonia solani,Corticium rolfsii, Phyophthora infestans, Sclerotz'nia scherotiorum,Xanthomonas oryzae, Cladosporium fulvum, so that these antibiotics ofthis invention are usable for agricultural fungicide or its components.They may be used in a form of powdery, liquid or granular compositionwith or without adjuvants or diluents. The concentration of theseantibiotics in such compositions may generally be about 0.0001 to about80% though the concentration may vary with the pests to be treated orwith the condition or others.

EXAMPLE 1 To an aqueous mixture (pH 7) containing 3% glucose, 2.2%soybean flour and 0.3% peptone is added 0.4% precipitated calciumcarbonate. 50 ml. aliquots of 4 liters of this mixture are measured into200 ml. Erlenmeyer flasks, which are then sterilized. Each flask isinoculated with 1 ml. of a seed culture of Streptomyces hygroscopicusvar. Iz'moneus (IFO-12703, ATCC No. 21431) which has been prepared byshake culture on a medium having the same composition as above for 2days, and incubated at 28 C. for 114 hours on a rotary shaker at 200r.p.m. The resulting broths are combined and filtered. The procedureyields 2.8 liters of a filtered broth. (This broth containing 200 'y/ml.of T-7545-A and 150 'y/ml. of T 7545B). Alternatively the culture ofStreptomyces hydroscopicus var. limoneus (IFO-12704, ATCC 21432) shows asimilar result.

EXAMPLE 2 To an aqueous mixture (pH 7) containing 3% glucose, 2.2%soybean flour and 0.3% peptone is added 0.4% precipitated calciumcarbonate. 500 ml. of aliquots of 2 liters of this mixture are measuredinto 4 Sakaguchis flasks of 2 liters-capacity, which are thensterilized. In each flask is inoculated with a loopful of a slantculture of Streptamyces hydroscopicus var. limoneus UFO-12703, ATCC No.21431). The inoculated flasks are incubated at 28 C. for 2 days, on areciprocating shaker having a svw'ng amplitude of 10 cm. at 120reciprocations/min. to prepare a seed culture. On the other hand, astainless-steel tank of 200 liters-capacity is charged with 100 litersof a medium prepared by adding 0.6% precipitated calcium carbonate to anaqueous mixture (pH 7) containing 5% glucose, 3.6=soybean flour and 0.5%peptone. The mixture in the tank is sterilized and, then, inoculatedwith 2 liters of the above mentioned seed culture and incubated at 27 C.for 114 hours under the condition of 50% aeration and 200 r.p.m. Theculture is filtered using as a filter aid 4 kg. of diatomaceous earthand 68 liters of a filtered broth containing a mixture of 100 'y/ml. ofT7545A and 150 'y/ml. of T7545B is obtained. Substantially similarresults are obtained when employing a culture of Streptomyceshydroscopicus var. limoneus UFO-12704, ATCC No. 21432).

EXAMPLE 3-A To 680 liters of a filtered broth (containing 100 'y/ml. ofT7545A, and 50 'y/ml. of T7545B) obtained as in the manner of Example 2,is added 13.6 kg. of active carbon. The mixture is stirred for one hourso that the active ingredients contained in the filtered broth isadsorbed on the active carbon. So-treated active carbon is thencollected by filtration, followed by washing twice with respective 136liter portions of water. So-washed active carbon is twice subjected toelution of the active ingredients with 136 liter portions of a mixtureof acetone-0.5 N hydrochloric acid (7:3), whereby 272 liters of theeluate is obtained, which is then passed through a column packed with 20liters of Amberlite lR-45 (OH-form). The passed solution is concentratedunder reduced pressure, followed by the addition of 10 times its volumeof acetone to obtain 4.2 kg. of a crude substance which contains 10 /mg.of T-7545-A and 15 'y/mg. of T-7545-B (Yield: ca. 62%).

EXAMPLE 3-B To 680 liters of a filtered broth (containing 100 'y/ml. ofT-7545-A, and 150 'y/ml. of T-7545-B) obtained as in the manner ofExample 2, is added 13.6 kg. of active carbon. The mixture is stirredfor one hour so that the active ingredients contained in the filteredbroth is adsorbed on the active carbon. So-treated active carbon is thencollected by filtration, followed by washing twice with respective 136liter portions of water. A column (70 liters) packed with so treatedactive carbon is subjected to elution with 250 liters of water saturatedwith butanol, so that the effective ingredient is eluted from the activecarbon. The eluate is concentrated under reduced pressure, and there isadded 10 times its volume of acetone to obtain 770 g. of a crudemixture, which contains 70 7/ mg. of T-7545-A and 100 7/ mg. ofT-7545-B. (Yield: ca.

EXAMPLE 4 The filtered broth obtained as in the manner of Exampie 2 (680liters containing 'y/ml. of T-7545-A and 150 'y/rnl. of T-7545-B) ispassed through a column packed with 55 liters of Amberlite IR- (H-form,S.V.=5), and then through another column packed with 65 liters ofAmberlite -IR-45 (OH form, S.V.=5). These columns are washed with 120liters of water, then the washings are combined with the passedsolution. The combined solution is passed (S.V.=2) through a columnpacked with' 60 liters of Dowex 'SOW-XZ (H form, 50- 100) mesh) to havethe active ingredients adsorbed on the resin.

After washing with 350 liters of water, the active ingredients areeluted with 200 liters of 0.5 N NH O'H. After the initial 30 liters ofeffluent is taken off. Then succeeding 125 liters of efliuent iscollected, and concentrated under reduced pressure, followed by theaddition of 10 times its volume of acetone to obtain 420 g. of powderycrude active ingredients, which contains ca. 150 /m-g. of T-7545-A and200-250 'y/mg. of T-7545-B (Yield: 92%).

EXAMPLE 420 g. of the crude powder obtained by Example 4 is dissolved in21 liters of pyridine-acetic acid buffer (0.1 M, pH 6.0). The solutionis poured at SVZ onto the column (17.8 x 146 cm.) packed with 35 litersof Dowex 50 X2 (50-100 mesh) bufierized with the same buffer solution asabove, whereby the active ingredients are adsorbed on the resin.

75 liters of the same buffer solution is further poured onto the column,so that impurities and pigments are eluted, followed by elution ofT-7545-B with 125 liters of pyridine acetic acid bulfer (0.1 M, pH 6.5)at SV2, then 75 liters of the eluate corresponding to 3-5 fractions (25liters/fraction) showing positive reaction to Anthrone reagent iscollected. So-collected eluate is concentrated under reduced pressure,followed by lyophilization, whereby 112 g. (750 'y/mg.) of crudeT-7545-B is obtained in a powdery form. The yield is ca. 90%.

Onto the column from which T-7545-B is eluted out, 500 liters ofpyridine, acetic acid buffer (0.1 M, pH 7.5) is poured at SV 2, then 250liters of the eluate corresponding to '5-l14 fractions (25liters/fraction) showing positive reaction to Anthrone reagent iscollected. So-collected eluate is concentrated under reduced pressure,followed by lyophilization, whereby 75 g. (750 'y/mg.) of crude T-7545-Ais obtained in a powdery form. The yield is ca. 90%.

EXAMPLE 6 42 g. taken out of the mixture (containing 150 'y/mg. ofT-7545-A and 200-250 'y/mg. of T-7545-B) obtained by Example 4, isdissolved in 210 ml. of water. The solution is passed through a columnpacked with 600 ml. of Amberlite IRA-900 (OH), (20-60 mesh), followed bythe successive elution with water to collect 200 ml. each of efiiuentfractionally.

The fractions containing the active ingredients positive to anthronereagent are detected, then the fractions are divided into T-7545-A-richfractions (3rd to 7th fractions) and T-7545-B-rich fractions (8th to16th fractions) by the aid of thin layer chromatography.

The former fractions (T-7545-A-rich fractions) are combined together,then a little amount of Amebrite IRC-SO (H) is added thereto, followedby the adjustment of pH to 8. The solution is concentrated under reducedpressure, then is added times its volume of acetone to give 5.6 g. ofcrude substance containing 650- 700 'y/mg. of T-7545-A and 250-300'y/mg. of T- 754'5-B.

The same procedure is conducted on the latter fractions (T-7545-B-richfractions) to give 9.5 g. of crude substance containing 200 'y/mg. ofT-7545-A and 600- 700 /mg. of T-7545-B. Yield ca. 85%.

'EXAMPLE 7 To 150 g. of active carbon for chromatograph grade(Tokusei-Shirasagi sold by Takeda Chemical Industries, Ltd, Japan) isadded 450 ml. of water, followed by dropwise addition of N HCl to makethe pH of the supernatant 3. The mixture is packed in a column of 750ml. (the ratios of its height to its diameter is 10), then the column iswashed with water to make the pH of the washing 3.5. Through this columnis passed at SV 3 a solution of 8.5 g. of crude T-7545-A (750 'y/mg.) in750 ml. of N/500 HCl to have the active ingredient adsorbed.

The column is washed with 3 liters of N/500 HCl, followed by the elutionof the active ingredient with mixture of methanol and N/500 HCl (3:7).The initial 0.5 liter of effluent is taken out, and is collected 2.0liters of the next efiluent which is colorless and positive to anthronereaction. The active fraction thus obtained is passed at SV 5 through acolumn (200 ml., the ratio of its height to diameter is 6) packed withAmberlite IR 45 (OH) which is previously washed with 30% aqueousmethanol to collect the passed solution. The column is washed with 500ml. of 30% aqueous methanol to wash out the active ingredients remainingin the column.

The passed solution is combined with the washing, and concentrated underreduced pressure. To the concentrate is added 10 times its volume ofacetone to obtain 5.4 g. of almost pure T-7545-A (950-1000 'y/mg.) aswhite powder (Yield: ca.

The same procedure as in the above on 8.5 g. of crude T-7545-B (750'y/mg.) obtained as in the manner of Example 5 gives 5.7 g. of almostpure T7545-B (950-1000 'y/mg.) as white powder (Yield: ca. 85%).

EXAMPLE 8 5.6 g. of T-7545-A-rich crude powder (700 'y/mg. T-7545-A, 300'y/mg. T-7545-B) obtained by the manner described in Example 6 isdissolved in 30' ml. of water. The solution is poured onto the column(ratio of height to diameter is 30), packed with 500 ml. of resins(Dowex 1-X2, OH) to have the active ingredients adsorbed on the resins.The resins are subjected to elution with water. The efiluent iscollected by 500 ml. each fraction, while examining the presence ofactive ingredients in each fraction by the aid of anthrone reagent,whereby T-7545-A is detected in the 5th-7th fractions and T-7545-B inthe 17th-25th fractions. The former fractions are concentrated underreduced pressure, followed by lyophilization, whereby 3.6 of T-7545-A(1000 'y/mg.) is obtained. The yield is about The latter fractions areconcentrated under reduced pressure, followed by lyophilization, whereby1.3 g. of T-7545-B (1000 'y/mg.) is obtained. The yield is about 80%.

9.5 g. of T-7545-B-rich crude powder (700 'y/mg. T-7545-B, 'y/mg.T-7545-A) obtained by the manner described in Example 6 is dissolved in50 ml. of water. The solution is poured onto the column (ratio of heightto diameter is 30) packed with 1 liter of resins (Dowex 1-X2, OH) tohave the active ingredients adsorbed on the resins. The resins aresubjected to elution with water. The effluent is collected by 500 ml.each fraction, while examining the presence of active ingredients ineach fraction by the aid of anthrone reagent, whereby T-7545-A isdetected in the 9th-l 1th fractions and T-7545-B in the 35th-60thfractions. The former fractions are concentrated under reduced pressure,followed by lyophilization, whereby 1.45 g. of T-7545-A (1000 /mg.) isobtained. The yield is about 85%. Same procedure on the latter fractionsgives 5.5 g. of T-7545-B (1000 'y/mg.). The yield is about 83%.

EXAMPLE 9 One gram taken out of A-rich crude mixture of T-7545-A andT-7545-B obtained by the above Example 6 is dissolved in 50 ml. ofmethanol. The solution is admixed thoroughly with 1 gram of silca-gel,and the mixture is dried under reduced pressure. The dried mixture isplaced on the top of the column (1.3 cm. X 90 cm.) packed withsilica-gel, followed by the column chromatography eluting withn-propanol:acetic acidzwater (4:1:1) by the aid of thin layerchromatography.

Then it is observed that T-7545-B is firstly eluted and then T-7545-A.The respective fractions containing each active ingredient are collectedand each of the fractions is subjected to concentration under reducedpressure, followed by the precipitation with acetone to obtain mg.

I of T-7545-B as white powder (Yield: ca. 65%) and 420 mg. of T-7545-Aas white powder (Yield: ca. 60%).

EXAMPLE In ml. of methanol is dissolved 1 g. of purified powder ofT-7545-A obtained by the manner described in Examples 7, 8 or 9. 2 ml.of N HCl is added to the solution, then water and the methanol aredistilled oif to obtain 1 g. of T-7545-A-hydrochloride.

The same procedure as above on 1 g. of purified powder of T-7545-Bobtained by the manner described in Example 7, 8 or 9 gives 1 g. ofT754SB hydrochloride.

Heretofore, organic arsenic agent has been widely used for the controlof the sheath blight of the rise plant, organic mercury compounds forthe control of stem rot, and pentachloronitrobenzene agent (hereinafterreferred to briefly as PCNB) for damping-off and sclerotial blight(southernblight) However, those materials are not always safe to men anddomestic animals or even to fish and plants. For instance, organicarsenic agent are not only chronically toxic to men and beasts but alsodetract from the yield of rice crop, while organic mercury compounds arenot free from chronic toxicity to men and animals, besides beingpoisonous to fish. PCNB is also injurious in that it adversely aifectsthe growth of young seedlings which are to be protected. In the light ofthe foregoing, development of a new improved drug has been seriouslyawaited.

The culture broth of a T-7545 producing strain of microorganism of thisinvention exhibits a powerful inhibitory action against sheath blight invivo test (application to young rice seedlings) despite the fact that itshows substantially no antimicrobial activity in ordinarymicrobiological assays (in vitro test). Further study has revealed thatthis antibiotic T-7545 has a peculiar characteristic that only when theplant or soil is treated therewith, does it show a strongdisease-controlling effect.

Furthermore, the antibiotic T-7545 has been found to be very efifectivenot only against the sheath blight of the rice plant but also againstother plant diseases such as the stem rot of rice and the damping-offand sclerotial blight of vegetables, [flowering plants and lumberseedlings. In addition, the antibiotic T-7545 is substantially harmlessto men and beasts, as well as to fish, and even when applied in a highconcentration, it does not substantially affect the germination, growth,yield and other features of useful plants.

The present economic poison composition may contain the culture broth ofan antibiotic T-7545 producing strain belonging to the actinomycetes,filtrate, thereof, the concentrate thereof, or a purified preparationthereof. It is also possible to employ antibiotic T-7545 in the form ofa free base or as the salts with suitable organic or inorganic acid(i.e. oxalic acid, succinic acid, sulfuric acid, hydrochloric acid,etc.) or with metals (e. g. sodium, cobalt, copper, aluminum, calcium,etc.). It is further permissible to employ an ester or ether of theantibiotic, for instance, which may be obtained by esterifying thehydroxyl of T-7545 with an acid (e.g. acetic acid, malonic acid or malicacid).

The compositions of this invention can be applied in any suitablemanner. Thus, depending upon the purpose of application, applicationtime, application method, etc., the composition may be directly appliedas such, after dissolution or dispersion in a suitable liquid carrier,or after blending with a suitable solid carrier. If desired, by addingan emulsifying agent, dispersant, suspending agent, adsorbent,penetrant, wetting agent, thickener, stabilizer or/ and adjuvant, thepresent composition may be used in such varied formulations as an oilsolution, emulsion, wettable powder, aqueous solution, soluble powder,dust, tablet, granule and spray, etc.

It is further allowable to use such a composition after compounding orblending with such other germicides as copper germicides, organic sulfurgermicides, organic chlorine germicides, organic phosphorus germicides,other antibiotics, etc., such insecticides as organic chlorineinsecticides, organic phosphorus insecticides, carbamic acidinsecticides, natural insecticides, etc., acaricides, nematocides,herbicides, plant growth regulators, synergistic agents, attractants,repellents, perfumes, plant nutrients, fertilizers and the like.

Toxicity of antibiotic substances T-'7545A and T- 7545-B are as follows:

(a) The toxicity of the antibiotic T-7545-A, which is one of theeifective ingredient of this composition, can be confirmed, forinstance, by administering the antibiotic T-7545A intravenously to miceand measuring the D value, or by measuring the median lethal dose forOryzias latipes. The results of such measurements are shown in Table 1.

another effective ingredient of this composition can be confirmed, forinstance, by administering the antibiotic T-7545-B intravenously to miceand measuring the LD value, or by measuring the median lethal dose forOryzias larzpes. The results of such measurements as shown in Table 2.

TABLE 2 Acute toxicity of T-7545-B Test animal Test procedure ToxicityMice Intravenous iniection LD 2,000 rug/kg. Oryzias latipes Leaving inwater solution. 'IL 40 p.p.m.

The concentrations of the active ingredient in the present fungicidesready for use is usually from about 0.0001% to about 1.0% by weight,more preferably about 0.0003% to about 0.3% by weight, in case of theliquid form (i.e. a solution, a suspension or an emulsion); while incase of the solid composition, from about 0.01% to about 30% by weight,about 0.1% to about 20% by weight being preferable. But, if necessary, acomposition containing a higher or lower concentration than theabovementioned value may be put into use. The content of the activeingredient of composition may be from about 0.5% to about by weightrelative to the composition, when it is prepared for a concentrate fonn.

EXPERIMENT l.RICE SHEATH BLIGHT CONTROL TEST Test procedure Test plants:Rice-Plants (variety: Kinmaze) were planted in earthen pots 9 cm. indiameter, 3 stocks per pot. Seedlings 80 days after potting were testedand 6 pots of them per group were used.

Inoculating method: Pathogenic fungi Pellz'cularia sasakii werecultivated on a plate of potato infusion-sugaragar at 30 C. for 48hours, and its agar disc 10 mm. in diameter was cut from a peripheralcolony. The disc was inserted into the inside space of the leaf sheathnear the soil surface, a disc per stem. The pots 'were kept in a PVCshelter at the atmospheric temperature of 32 -2S C. and the relativehumidity of -70%. Inoculation was carried out immediately after theapplied drug has been air-dried in the prophylactic test, and 3 daysbefore application in the remedial test.

Application of the drug: A series of aqueous solutions prepared from a10,000 dilution units/ml. cultural broth of Streptomyces hygroscopicusvar. limoneus (IFO 12704, ATCC No. 21431) and diluted liquidpreparations of commercial active control drugs were prepared. To eachof those solutions and preparations, 0.05% (final concentration) of Dyne(a spreader manufactured by Takeda Chemical Industries, Ltd.) was added,and the mixture was sprayed evenly over the foliage by means of aspraygun at the rate of 30 ml. per 6 pots. The cultivation of theantibiotic-producing strain was carried out in the following manner. 60ml. aliquots of a liquid medium were measured into 200 ml. Erlenmyerflasks, and each fiask was inoculated with 1 loop of the above strain.The flasks were incubated at 28 C. for 5 days on a rotary shaker at 200r.p.m.

The above medium had the following composition. 3% glucose, 2.2% rawsoybean powder, 0.3% peptone and 0.4% calcium carbonate.

Assay method: 10 days after application of the drug, the length of eachstem from ground level to the upper edge of the lesion was directlymeasured.

Result TABLE 3 Sheath blight control effect of the cultural broth (Theaverage length of lesions/stem (cm.))

Preven- Reme- Injury tlve dial to Drug Dosage effect efiect plantUntreated control 27.9 30.1 Polynxlne PB emulslflable concentrate X6006.4 15.0 Monzet wettable powder X2,000 1.1

- 0 0.4 Cultural broth of Streptomyces hyg I groacopicus var. limomus(IFO X8 0 12704, ATCC No. 21431) X16 0 L X32 0 3.7

1 Containing 30,000 PS units/g. as polyoxlne B (see note).

Containing 20% of methylarsine-bis-dlmethyldlthlocarbamate, 20% ofzinc-d1methyl-dlthiocarbamate and 40% of his- (dimethyl-thlocarbamoyl)disultlde.

3 Undiluted.

NOTE.-PS represents Pelltoulana sasakii and PS units/g. as polyoxine Bmeans the units/g. of the etfective fraction of polyoxln B against PS.

Evaluation Up to a dilution multiple of 16, the cultural broth washighly effective, in terms of both prophylaxis and treatment, againstthe sheath blight of rice, and no injury at all to the plant wasobserved.

Experiment 2.-Rice sheath blight control test by pretreatment of thesoil.

Test procedure Test rice plants: Same as Experiment 1.

Inoculating method: Same as above.

Application of the drug: Aqueous solutions of a T-7545-A powder weresprayed as per the method of Experiment 1 or soil-injected at the rateof 5 ml. per pot.

Assay method: As per Experiment 1.

T-7545-A was as effective against the sheath blight of rice bysoil-injection as by spraying.

No injury to the plant was observed, either, in preemcrgence treatment.

18 EXPERIMENT 3.-RICE SHEATH BLIGHT CONTROL TEST Test procedure Testplants: Rice plants (variety: Kinmaze) were planted in a/5,000 Wagnerpots, 3 stocks/pot. 5 pots per group, in the ear-bearing stage were usedfor test.

Inoculating method: According to Experiment 1, an agar disc 20 mm. indiameter was fitted on the leaf sheath near the soil surface, 1 disc/2stems, and the pots were placed in a vinyl house.

Application of the drug: 5 days after inoculation T-7545-A was appliedat the dosage of ml./5 pots as per Experiment 1.

Assay method: 21 and 35 days, respectively, after treatment, theprocedure of Experiment 1 was followed.

At the concentration of 10 p.p.m. or higher T-7545-A was highlyeflective in the treatment after the disease emergency, and suppressedthe formation of new lesions for more than a month after exposure. Noinjury whatever, was observed even at the concentration of 40 p.p.m.

Experiment 4.-Rice stem rot control test Test procedure Test plants: Asper Experiment 3.

Application of the drug: Same as above.

Inoculating method: Pathogenic fungi Helminthosporium sigmoideum werecultured on rice-straw medium at 28 C. for 14 days/piece (2 cm.length)/stem of the resulting culture was fitted into the inner space ofthe bottom leaf sheath of each rice plant stem, after the sprayed drughad been air-dried. Thereafter, the procedure as per Experiment 1 areemployed and the pots were placed in a vinyl house.

Assay method: 14 days after inoculation, the length of the lesion ineach stem was measured.

Result TABLE 6 Stem rot control efiect ol T-7545EA (the average lengthof lesions/stem cm.

Preven- Injury tive to Drug Dosage efliect plant Untreated control 8. 5X2 500 2.1 Fumiron wettable powder xlzzm 0 6 i 3 10 2.3 T-7545-A 9 201.8 40 0.4

1 Containing 5% of phenyl mercuric iodide:

9 Parts per million:

Evaluation At the concentration of 10 p.p.m. or higher, T-7545-A showeda strong effect upon stem rot.

Experiment 5.Cucumber damping-off control test Test procedureRhizoctonia solani was cultured on barley medium at 28 C. for 5 days. Afield soil sample was packed into 19 earthen pots 9 cm. in diameter,which were then sterilized with steam. The chaff inoculum was evenlyworked into the cover soil of each pot to the depth of about 3 cm., atthe rate of 2 g./pot. The pots were kept in an inoculation chamber at 28C. for 24 hours and, then, transferred to a greenhouse.

Application of the drug: As per Experiment 1, the drug was sprayed ontothe soil surface at the rate of 30 ml./ 6 pots/ group.

Test plant: Healthy seeds of cucumber (variety: Suyo) were sown byburial in the pots immediately after treatment, at the rate of 10seeds/pot. The pots were then placed in a greenhouse at 32 C.- 28 C.

Assay method: 14 days after sowing, the results were evaluated againstthe following coeflicients of lesion and the degrees of damage werecalculated.

Coefiicients of lesion (1):

: Healthy 0.5: Root hairs alone are slightly effected.

1: Aerial portions near soil surface and roots effected. 2: Early stageof damping-oil.

3: Aifected at germination; growth inhibited.

wherein n means the number of samples corresponding to each coefiicientof lesion.

Result Degree of damage (percent) X100 TABLE 7 Damping-on control effectof T-7545-A Degree of Injury damage to Drug Dosage (percent) plantUntreated control 100 Pentachloronitrobenzene 10 50% wettable powder(control) l 5 g 1 Parts per million.

Evaluation At the concentration of 10 ppm. or higher, T-7545-A washighly effective against the damping-01f caused by Rhizoctonia solaniand had no injurious effect upon seed germination and the growth ofseedlings.

Experiment 6.Cucumber damping-off control test, by seed coating Testprocedure Inoculating method: As per Experiment 5.

Application of the drug: Seeds were wetted with water and, then, lightlycoated with a T-7545-A powder.

Average deposition rate: 80 mg./ 10 seeds.

Test plant: As per Experiment 5.

Assay method: Same as above.

1 Powdereeoating with adjuvant (talc) only.

Evaluation T-7545-A is also effective against damping-off when appliedas seed dust coating. In this method, no injurious effect was observed,either.

Experiment 7.-Cucumber Southern blight control test (Test procedure)inoculating method; As per Experiment 5, pathogenic fungi Corticiumrolfsii were cultured for 10 days and the resulting culture was workedinto the soil at the rate of 5 g. per pot. The pots were kept aspreviously explained.

Application of the drug: As per Experiment 5, the drug was applied atthe rate of 30 ml./6 pots/ group.

Test plants: Same as Experiment 5.

Assay method: Same as above.

Result TABLE 9 Southern blight control efiect of T-7545-A At theconcentration of 10 p.p.m. or higher, T-7545-A was highly efiectiveagainst Southern blight and not injurious to the plant.

Experiment 8.Cucumber powdery mildew control test Test procedure Testplant: Healthy cucumber (variety: Suyo) planted in the a/5000 Wagnerpots. The 3 pots group of seedlings 60 days after potting were used.

Inoculating method: The above pots and the another pots planted cucumberwhich have already infected by Sphaerotheca fuliginea are stood oneafter the other so as to be taken ill by the natural infectionrespectively.

Application of the drug: A drug solution prepared in the same manner ofExperiment 2 is applied at the rate of ml. per 3 pots.

Assay method: Sample leaves which were healthy before the application ofthe drug but then have taken ill, are employed for this assay. The areaof the lesion of 4 leaves per pot was measured.

Result TABLE 10 Cucumber powdery mildew control efiect of T-7545-A (theaverag specific area of lesions/leaf (percent)) 1 Containing 19.5? of 2-l-meth lhe t 1-46-dinitro hen l 9 Parts p'er million i y p y) p y MommaEvaluation T-7545-A was effective as a control for powdery mildew,having no injurious etfect upon cucumbers.

Experiment 9.--Tomato late blight control test Test procedure Testplants: Tomato plants (variety: ponterosa) are planted in l2-cm. indiameter pots, one plant per pot.

21 The seedlings 50 days after potting were used, 6 pots/ group.

Application of the drug: Applied at the rate of 100 ml./ 6 pots/ groupas per Experiment 2.

Inoculating method: Pathogenic fungi (Phytophthora infestans) werecultured on a potato infusion-agar plate at 20 C. for 10 days and theresulting culture was used to prepare a zoospore water suspension (about10 zoospores/ml.). One day after the application of the drug, the plantswere spray-inoculated with 20 ml. of the suspension per pot. The potswere kept in an inoculation chamher at 20 C. for 24 hours and, then,transferred to a greenhouse.

Assay method: 7 days after inoculation, the area of each lesion wasmeasured.

Result TABLE 11 Tomato late light control effect or T-7545-A Percentratio 1 Containing 75% of tetrachloroisophthalouitrile (Active controldrug commonly used).

2 Parts per million.

Evaluation T-7545-A was effective against the late blight of the tomatoplant, showing no injurious eyect on the plant.

Experiment 10.-Kidney bean stern rot control test Test procedure Testplants: One Kidney bean plant (variety: Taisho Kintoki) planted in eachof 12 cm. diameter pots; the completely unfolded uppermost leaves cutoff 40 days after potting were used, 6 leaves/ group.

Application of the drug: To each solution of the drug, spreader Dyne wasadded until 0.05% (final conc.), and the above leaves were dipped andimmediately allowed to air-dry.

Test vessel: 10 ml. water was put in Petri dishes 9 cm. in diameter, anda polyvinyl-chloride ring was placed in each of those humid vessels. Thetreated leaves were placed on top of the rings, a leaf per ring.

Inoculating method: Pathogenic fungi Sclerotinz'a sclerotiorum werecultured on a potato infusion sugar plate at 20 C. for days, at the endof culture time some agar disc 10 mm. in diameter were cutout from aperipheral colony. Each one disc was placed on the center of each leafand, then, kept in a thermostatic vessel at C.

Assay method: 6 days after inoculation, the area of the lesion in eachleaf was measured.

1 Parts per million.

2 gontalning 50% of 2,6-diehloro-4-nitroaniline (control drug commonlyuse Evaluation T-7545-A was effective against the stem rot of Kidneybean, and had no injurious elfect upon the plant.

Experiment 11.-Pests control test with compound dusts Test procedureSheath blight: As per Experiment 1, compound dusts were applied using acompact fine-duster at the rate of 300 mg. to 6 pots, 3 days afterinoculation.

Bacterial leaf blight: Same as above, except that the seedlings 25 daysafter potting were treated.

Pathogenic bacteria X anthomonas oryzae were cultured on asucrose-bouillon medium at 28 C. for 1 day, and the uppermost leaf ofeach seedling was stabbed with a needle carrying a suspension 10 /1111.of the bacterial cells. The pots were kept in an inoculation chamber at24 C. for 24 hours, and, then the pots were transferred to a greenhouse.2 days after the inoculation, the pots were dusted in the same manner asabove.

The length of each lesion was measured 10 days after inoculation.

Blast: Same as bacterial leaf blight.

Pathogenic fungi Pyricularia oryzae were cultured on a rice-strawinfusion-agar medium at 28 C. for 10 days, and the pots werespray-inoculated with a suspension of the conidia (10 /ml.) at the rateof 2 ml. per pot. The pots were kept in an inoculation chamber at 28 C.for 24 hours. The pots taken out from the box were dusted in the samemanner as above and, then, transferred to a greenhouse. 7 days after theinoculation, the specific ratio of the average area of lesion wasmeasured by Okamotos method.

Rice stem borer: Rice plants in a./ 5,000 Wagner pots, 60 days afterpotting and on the fourth day of penetration by the larvae of Chilesuppressalis were dusted at the rate of 5 g. to 5 pots.

On the 8th day of penetration, the stems were dissected and the survivalrate of the larvae was calculated.

Green rice leafhop-per: Immediately after dusting in the same manner asdescribed above for Rice stem borer, the rice plants were covered withnetting, and adultleafhoppers (Naphottetix cinctz'ceps) were set free inthe netting.

After one day, the result was examined and the survival rate calculated.

Evaluation The all mixed formulations showed no reduction in actionagainst shealth blight and each used pests, and none of them had aninjurious effect upon the rice plants.

Results The control effects of various mixed formulations were astabulated below.

1 Percentage (percent) with untreated control as 100.

Test procedure Test plants: As per Experiment 9.

Application of the drug: Same as above.

Inoculating method: Pathogenic fungi Cladosporium fulvam were culturedon a potato infusion-sucrose plate at 20 C. for 14 days, and asuspension of conidia (10 ml.) was prepared from the culture.

The plants were spray-inoculated with the suspension 3 days beforeapplication of the drug, at the rate of 5 ml. per pot. The pots werekept in an inoculation chamber at 25 C. for 24 hours, and, then theywere transferred to a greenhouse.

Assay method: 14 days after inoculation, the specific ratio of theaverage area of lesion in the leaves was T-7545-A was effective aaginstthe tomato leaf mold, and had no injurious effect upon the plants.

TABLE 14 Sheath blight, Blast percent specific T-7545A Percent lesionarea of concenconcenenlargelesion 1 5 tration Ingredient mixed trationment 1 0.3 Blastlcidin-S-benzyl 0. 2 0. 3

erninobenzene- 7 ii 0 2 10 0 a 0.3 o

a Kasugamycin- 0.2 0.4 10

0.3% -.do 0.2 8 0.3 0,0-diisopropyl-S- 1. 5 2. 7

benzylthiophosphate. 0.3% do 1. 5 5 2. 5

O-ethyl-S,S-diphenyl- 2 0. 3

dithiophosphate. 0.3% do 2 3 0. 2

1 Percentage (percent) with untreated control as 100.

TABLE 15 Sheath blight, Percent T-7545-A Concenpercent survivalconcentratration, enlargerate of tion Ingredient mixed percent ment 1rice stern ls: borer 0.27 12 5 0 1,3-bis(carbamoyl- 2 Ithio)-2-(N,N-dimethy1amino)- propane hydrochloride. 0.2% .do 2 8 0Dimethyl(3-methyl- 2 3 l-nitrophenyl) thiephosphate. 0.2% d0 2 9 3 1Percentage (percent) with untreated control as 100.

TABLE 16 Sheath Percent blight, survival T-75i5 Coneenpercent rate ofconcentratration, lesion geen rice tion Ingredient mixed percentexpansion leathopper l-napht-hyl-N-methyl- 1. 5 4

carbamate. 0.3% do 1. 5 7

1 Percentage (percent) with untreated control as 100.

Experiment l2.Tomato leaf mold control test o 24 Experiment ISL-Ricesheath blight control test.

Test procedure Test plants: As per Experiment 3. inoculating method: Asper Experiment 3.

At the concentration of 20 ppm. or higher, T-7545-B was highly effectivein the treatment after the disease emergency, and suppressed theformation of new lesions for more than two weeks after exposure. Noinjury whatever, was observed even at the concentration of p.p.m.

Experiment 14.Rice sheath blight control test for the mixture ofantibiotics T-7545-A and T--7545B Test procedure Test plants: As perExperiment 1.

Inoculating method: As per Experiment 1.

Application of the drug: As per Experiment 3 except that T-7545-A isexchanged for T-7545-A and T- 7545-B, or a mixture thereof.

Assay method: As per Experiment 1.

Results TABLE 19 Sheath blight control eficct of a mixture of T-7545-Aand T-7545-B Percent 01 average length of diseased region Dosage of drug(p.p.m.) (percent) Untreated control 1 T-7545-A (2.5 p.p.m.) 23 T-7545-A5 p.p.m.) 11 T-7545-B 6.25 p.p.m.) 65 T-7545-B (12.5 ppm.) 47 T-7545A(2.5 p.p.m.)+T-7545-B (6 1 T-7545-A (5 p.p.n1.)+T-7545B (12.5 p.p.m.) 1

TABLE 20 Sheath blight control efiect of a mixture of .T7545-A andT-7545-B Percent of average length oi diseased region Dosage of drug(p.p.m.) (percent) Untreated control 1 100 T-7545-A (5) 25 T-7545-A(10)- 21 T-7545A 2 'I7545B (10)- 76 T-7545-B (20)- 7 T-7545B (40) 4T-7545A(8)+ 5 T7545A(5) +T-7545-B (5) 5 'I7545-A(2) +T-7545-B (8)- 42Evaluation TABLE 21 Damping-oi? control efieet of T-7545-B Degree 01'damage (percent) Drug Dosage Untreated control PentachloronitrobenzeneX1,000 50% wettable powder (control) X500 to H HNIOUIOD 1 Parts permillion.

Evaluation At the concentration of 20 p.-p.m. or higher, T-7545-B washighly effective against the damping-off caused by Rhizoctonia and hadno injurious eifect upon seed germination and the growth of seedlings.

Experiment 16.Cucumber damping-oil control test, by seed coating Testprocedure Inoculating method: As per Experiment 15. Application of thedrug: Same as above. Test plant: As per Experiment 15. Assay method.Same as above.

Result TABLE 22 Dsmplng-ofi control efiect of T-7545-B, by seed coatingPentaehloronltro T-7545-B dust benzene dust Degree 0! Injur De ee ofInur Contents 01 eflective tngredamage to y d a mage to y dlent(percent) (percent) plant (percent) plant Powder-coating with adjuvant(talc) only.

Evaluation T-7545-B is also eflective against damping-off when appliedas seed dust coating. In this method, no injurious efiect was observed,either.

Experiment l7.Cucurnber damping-01f control test for a mixture ofT7545-A and T-7545-B Test procedure Rhizactonia solani was cultured onbarley medium at 28 C. for 5 days. In 10 kg. of the field soilsamplewhich were sterilized with steam, is admixed 1.2 kg. of the cultivatedchaif, and was Worked into the cover soil to the total of about 5 cm.and then it stand for 24 hours under the 100% relative humidity at 28 C.And then, the mixture is dried at room temperature for 2 days and thecultivated chaif is passed through in the sieve and filtered 01?. Theinoculated soil of Rhizoctonia solani obtained were kept into earthenpots 12 cm. in diameter at 450 g./pot.

Application of the drug: As per Experiment 5, the drug was sprayed ontothe soil surface at the rate of 40 ml./2 pots/group.

Assay method: As per Experiment 5.

Results TABLE 23 Cucumber damping-on contro egzgtor a mixture ofT-7545-A and Percent of Content of effective ingredient (p.p.m.) damage(percent) Untreated control 12g 24 11 88 32 16 56 16 3Pentachloronltrobenzene (500)... 16 Pentachloronltrobenzene (1,000) 3TABLE 24 Cucumber damping-0d control efiect Percent of survival seedingtrom damping Content of Concentration Germi- 06 after efiectiveapplication nated 14 days ingredient method seed (percent) Untreatedcontrol 0 0 Uninnnnlaflnn 90 90 5 p.p.m. use of soil 20 16 13 p.p.m. useof soil... 63 53 26 p.p.rn. use of soil- 88 82 T4545 0.625% powder seedcoa 63 12 1.25% powder seed coat 75 37 2.5% powder seed coat 97 83 5.0%powder seed coat- 95 10% powder seed coat- 92 92 Evaluation T-7545-B washighly effective in the cucumber damping-off control eifect,particularly, a mixture of T-7545-B and T-7545-A have shown the higheifectiveness of involution.

Experiment 18.Rice sheath blight control test Test procedure Testplants: Rice. As per Experiment 3.

Inoculating method: As per Experiment 3.

Application of the drug: As per Experiment 3.

Assay method: As per Experiment 3, except to rain artiiically from theunnatural-rain-tower at 25 mm. per 30 minutes, after one hour fromapplication of the drug.

1 Parts per million.

Experiment 19.-Rice sheath blight control field test Test procedure Testplace: Fukuchiyama, Japan.

Test plants: Rice plant (variety: Gohyakumankoku) which were planted inpaddy in May 1, and seedling in July, 18, were used this test.

inoculating method: Pathogenic fungi Pellicularia sasaki were culturedon rice-straw 2 cm. length medium at 28 C. for 5 days. These straws wereinserted into the inside space of leaf sheath near the paddy field waterin July 4.

Application of the drug: Each drug was sprayed evenly over the ricefield by means of a spray-gun at the rate of 200 liters/ 10 a. (aqueoussolution) and 3 kg./ 10 a. (dust) in July 11 and July 17, respectively.

Assay method: The results were evaluated against the followingcoefiicients of lesion of each stem and the degree of damage werecalculated.

Coefiicients of lesion:

3: Formation of disease to flag leaf sheath 2: Formation of disease tosecondary leaf sheath 1: Formation of disease to 3-4 leaf sheath Degreeof damage 3 (stem number indicated 3) +2 (stem Result TABLE 26 Averagelength of Percent of Coneendiseased disease Content of efieetlveingredient; tration region (0111.) stem Untreated control 88. 2 100.Polyoxcin PS emulsifiable con- X600 48. 0 84. 4 centrete (3%) X600 48. 084. 4 Monkit solution X2, 000 19. 8 41. l T-7545-A-S0l11t-ion. 2 28 19.2 45. 2 T-7545-A-dl18t 3 0. 28 16. 8 40. 3

l Containing 6.5% of iron ammonium-methanearsonie acid. 8 Parts permillion. 8 Percent.

EXAMPLE 1 Wettable powder: Percent T-7545-A 1.0 Sodium ligninsulfonate0:1 Polyoxyethylene alkylarylether 0.1 White carbon 0.1 Clay 98.7

Depending upon the purpose and method of application, the aboveformulation is diluted to the range of 1-200 ppm. in terms of thepresent antibiotic and the diluted preparation is sprayed by means of asprayer, applied to the soil surface, or above powder is usedundilutedly to coat the seed.

EXAMPLE 2 Tablets: Percent T-7545-A hydrochloride 15.0 Polyoxyethylenealkylarylether 2.0 Lactose 83.0

Before application, the above formulation is diluted to The aboveformulation is diluted to the range of 500- 100,000 p.p.m. and thediluted preparation is sprayed by means of a fine concentrate sprayerfrom aircraft, for instance.

EXAMPLE 4 Emulsi'fiable concentrate: Percent T-7545-A 10.0Polyoxyethylene alkylarylether 5.0 Methanol 20.0 Methylnaphthalene 40.0

Dimethylformamide 25.0

28 Before application, the above formulation is diluted to theconcentration range given in Example 1.

Depending upon the objective and mode of application, the above mixtureas such is dusted by means of a number indicated 2) 1X (stem numberindicated 1) X 100 3 X all test stem duster at the rate of l8 kg. to 10a., or used to coat the seed.

EXAMPLE 6 Mixed dust A: Percent T-7545-A 0.2 2-amino-1,3,4-thiadiazole-5-thiol 5.0 Aluminum stearate 0.02 Talc 94.78

The above preparation is applied according to Example 5.

EXAMPLE 7 Mixed dust B: Percent T-7545-A 0.3Z-formamido-l,3,4-thiadiazole 4.0 Talc 95.7

The above preparation is applied as per Example 5.

EXAMPLE 8 Mixed dust C: Percent T-7545-A 0.2 2-amino-1,3,4-thiadiazole3.0 Talc 96.8

The above preparation is applied as per Example 5.

EXAMPLE 9 Mixed dust D: Percent T7545A 0.3 3 benzylideneamino 4phenylthiazoline 2- thione 5.0 Talc 94.7

The above preparation is applied as per Example 5.

EXAMPLE 10 Mixed dust E: Percent T-7545A 0.3 BlasticidinS-benzylaminobenzenesulfonate 0.2 Talc 99.5

The above preparation is applied as per Example 5.

EXAMPLE 11 Mixed dust F: Percent T-7545-A 0.3 Kasugarnycin 0.2 Talc 99.5

The above preparation is applied as per Example 5.

EXAMPLE 12 Mixed dust G: Percent T-7545-A 0.30,0-diisopropyl-S-benzylthiophosphate 1.5 Talc t- 98.2

The above preparation is applied as per Example 5.

1,3 bis(carbamoylthio) 2(N,N dimethylamino) propane hydrochloride 2.0Talc 97.8

The above preparation is applied as per Example 5.

EXAMPLE 16 Mixed dust K: Percent T-7545-A 0.2 Dimethyl(3 methyl4-nitrophenyl)thiophosphate. 2.0 Talc 97.8

The above preparation is applied as per Example 5.

EXAMPLE 17 Mixed dust L: Percent T-7545-A 0.3l-naphthyl-N-methylcarbamate 1.5 Talc 98.2

The above preparation is applied as per Example 5.

EXAMPLE 18 Wettable powder: Percent T-7545-B 1.0 Sodium ligninsulfonate0.1 Polyoxyethylene alkylarylether 0.1 White carbon 0.1 Clay 98.7

Depending upon the purpose and method of application, the abovefomulation is diluted to the range of 2-400 p.p.m. in terms of thepresent antibiotic and the diluted preparation is sprayed by means of asprayer, applied to the soil surface, or above powder is usedundilutedly to coat the seed.

30 EXAMPLE 21 Emulsifiable concentrate: Percent T-7545-B 10.0Polyoxyethylene alkylarylether 5.0 Methanol 20.0 Methylnaphthalene 40.0Dimethylformamide 25.0

Before application, the above formulation is diluted to theconcentration range given in Example 18.

EXAMPLE 22 Dust: Percent T-7545-B 0.2 Aluminum stearate 0.02 Talc 99.78

Depending upon the objective and mode of application, the above mixtureas such is dusted by means of a duster at the rate of 1-8 kg. to 10 a.,or used to coat the seed.

The above preparation is applied as per Example 5.

EXAMPLE 25 Mixed dust 0: Percent T-7545-B 0.4 Kasugamycin 0.2 Tale 99.4

The above preparation is applied as per Example 5.

EXAMPLE 26 Mixed dust P: Percent T-7545-B 0.40,0-diisopropyl-S-benzylthiophosphate 1.5 Talc 98.1

The above preparation is applied as per Example 5.

EXAMPLE 27 Mixed dust Q: Percent T-7545-B 0.4 O-ethyl-S,S-diphenyldithiophosphate 2.0 Talc 97.6

The above preparation is applied as per Example 5.

EXAMPLE 28 Mixed dust R: Percent T-7545-B 0.2 Polyoxine (B; 1,000 PSunits/g.) 0.1 Talc 99.7

The above preparation is applied as per Example 5.

EXAMPLE 29 Mixed dust S: Percent T-7545-B 0.4 1,3-bis(carbamoylthio) 2(N,N dimethylamino)propane hydrochloride Talc 97.6

The above preparation is applied as per Example 5.

31 EXAMPLE 30 Mixed dust T: Percent T-7545-B 0.3 Dimethyl(3-methyl 4nitrophenyl)thiophosphate 2.0 Talc 97.7

The above preparation is applied as per Example 5.

EXAMPLE 31 Mixed dust U: Percent l-naphthyl-N-methylcarbamate 1.5 Talc98.1

The above preparation is applied as per Example 5.

EXAMPLE 32 Mixed dust V: Percent Talc 99.1

The above preparation is applied as per Example 5.

EXAMPLE 33 Mixed dust W: Freeze-dried powder of the culture broth ofStreptomyces hygroscapicus var. limoneous (lFO 12703, ATCC No. 21431):100% The above preparation is applied as per Example 5.

What is claimed is:

1. A member selected from the group consisting of antibiotics T-7S45-Aand T-7545-B and a salt thereof with hydrochloric, sulfuric, oxalic orsucciuic acid, wherein T-754S-A has the following properties:

(a) A weakly basic white hygroscopic powder, which has no definitemelting point, and decomposes at 100135 0.;

( b) Readily soluble in water and polar organic solvents,

sparingly soluble in acetone and ethanol, and insoluble in ethylacetate, ether and petroleum ether;

() Color reactions are as follows:

Molish reaction: positive Fehling reaction (under heating): positiveAnthrone reaction: positive Phenol-sulfuric acid reaction: positiveOrcinol-sulfuric acid reaction: positive Naphthoresorcin-sulfuric acidreaction: positive Benzidine-periodate reaction: positivePeptide-detection-reagent reaction (tertiary butylhypochloride):positive Alkaline potassium permanganate: reduce Ninhydrin reaction:faint purple color (d) pKa value is 6.2;

(e) The molecular weight estimated by titration is (f) No characteristicabsorption is observed at above (g) Significant infrared absorptionbands are: 3450(), 2900(M), 1640(M), 1450(M), 1410(M), 1370(M), 1160(M),1200-1000(8), 920(W), 900(M), 8550M),

The abbreviations M, W and S in parentheses denote medium absorptions,weak absorptions and strong absorptions, respectively;

(b) Optical rotation: [a] =110il5 (c.=1, H O);

110il5 (c.=l, pyridine); 92.5:10 (c.= 1, dimethylformarnide) (i)Elementary analysis: C, 47.6115 H, 7.17i0.5%;

(j) Rf values on paper partition chromatogram and on silica gelthin-layer chromatogram:

Thin-layer chroma- Paper chromatography tography hydro- Solveut systemT-7545-A chloride T-7545-A n-Butanolzacetic acidrwater (4:1:2)- 0. 10-0.12 0. 10-0. 12 0. 10 70% aqueous acetone 0. 44 0. 37-0. 44 0. 53 n-Butauol: ethanol: water: cone.

aqueous ammonia (402102491)--- 0. 11-0. 12 0. 11-0. 12 0. 08

Ethyl aeetatezacetic acid: water :1: 0.01 0. 01 0.00 Ethylacetatmpyridinezwater (2:1: 0 01-0. 02 0. 01-0. 02 0.01n-Butanolzpyrldinezwater (412:1).-- 0. 0.05 0. 18 n-Butanolzpryidlnezwater (4 :7) 0. 46-0. 48 0. 46-0. 48 0. 27 phenol (N H;)0.55 O. 55 0. 38 11-13 utanol: ethanol water pyridine (35:15:40z10) 035-0. 39 0. -0. 39 0. 44 n-Propanolzwaterzeonc. aqueous ammonia(70:29zl) 0. 06-0. 07 0. 06 0. 07 n-Propanoltacetic acidzwater (k) Paperelectrophoresis using boric acid butter (pH 10) at a potentialdifference of 2 kv. for 2 hours: +1.5 cm.;

Paper electrophoresis using pyridine-acetic acid (pH 6.0) at a potentialdifference of 2 kv. for 3 hours: 8.5 cm.;

and T-7545-B has the following properties:

(a) A weakly basic white hygroscopic powder, which has no definitemelting point, and decomposes at'95 to C.

(b) Readily soluble in water, polar organic solvents;

sparingly soluble in acetone, and ethanol; and insoluble in ethylacetate, ether and petroleum ether;

(c) Color reactions are as follows:

Molish reaction: positive Fehling reaction (under heating): positiveAnthrone reaction: positive Phenol-sulfuric acid reaction: positiveOrcinol-sulfuric acid reaction: positive Naphthoresorcin-sulfuric acidreaction: positive Benzidine-periodate reaction: positivePeptide-detection-reagent reaction (tertiary butylhypochloride):positive Alkaline potassium permanganate: reduce Ninhydrin reaction:faint purple color (d) pK'a value is 5.0

(e) The molecular weight estimated by titration is (f) No characteristicabsorption is observed at above 210 III/L;

(g) Significant infrared absorption bands are: 3400(8), 2910(M),1638(M), 1415(M), 1080(8), 1025(8), 900(M), 840(M);

The abbreviations M, W and S in parentheses denote medium absorptions,weak absorptions and strong absorptions, respectively;

(h) Optical rotation [a] =l02i1O (c.'=1, H O) (i) Elementary analysis:C, 46.46i1.5%;

7.06i0.5%; N, 2.44J -0.5%

(j) R values on spot films for silica gel thin-layer chromatographicuse: 0.43 (n-propanolzacetic acid: water: 4:1:1)

(k) Electrophoresis paper electrophoresis using boric acid butter (pH10) at a potential difierence of 2 kv. for 2 hours: +6.0 cm.;

paper electrophoresis using pyridine-acetic acid (pH 6.0) at a potentialdifierence of 2 kv. for 3 hours: -4.0 cm.

2. A compound as in claim 1, wherein the acid salt is hydrochloride orsulfate.

3. A process for producing an antibiotic T-7545, which comprisescultivating, by a surface culture method or submerged culture method,Szreptomyces hygroscopicus var. limoneus ATCC No. 21431 or ATCC No.21432 in a culture medium containing an assimilable carbon source, adigestible nitrogen source and an inorganic salt at a temperature of 15to 45 C. and a pH of 5 to 10, until the antibiotic has accumulated inthe culture medium.

4. A process for producing an antibiotic T-7545, which comprisescultivating, by a surface culture method or submerged culture method,Streptomyces hygroscopz'cus var. limoneus ATCC No. 21431 or ATCC No.21432 in a cul ture medium containing an assimilable carbon source, a

34 digestible nitrogen source and an inorganic salt at a temperature of15 to 45 C. and a pH of 5 to 10, until the antibiotic has accumulated inthe culture medium, and recovering the antibiotic from the resultingbroth.

References Cited Miller, The Pfizer Handbook of Microbial Metabolites,

McGraw-Hill Book Co., Inc., New York, N.Y., 1961, p. 580.

JEROME v. GOLDBERG, Primary Examiner U.S. Cl. X.R. l95--80

